Sensorineural hearing loss is most commonly a result of hair cell degeneration. Hair cells in the organ of Corti cannot be replaced once lost, and therefore, sensorineural hearing loss is permanent. The bHLH transcription factor Math 1 has been shown to specify the embryonic development of hair cells and to induce growth of extranumerary hair cells in explants of rat inner ears. Using in vivo inoculation of an adenovirus vector with the Mathl gene insert into the endolymph of the mature pre-deafened guinea pig cochlea, we were able to generate new cochlear hair cells in the organ of Corti. We have shown that non-sensory cells in the mature cochlea retain the competence to generate new hair cells upon over-expression of Mathl in vivo, and that Mathl is sufficient to direct hair cell differentiation in these mature non-sensory cells. We have also demonstrated that new cochlear hair cells attract auditory neurons and that ABR-measured auditory thresholds significantly improve due to the Mathl treatment in deaf animals. In this application we propose to further characterize the structural and functional outcome of Mathl -induced hair cell regeneration in the mature deaf cochlea. Our first Aim is to determine the extent of hair cell regeneration following Mathl over expression in cochleae deafened with severe ototoxic insults, and to characterize the new hair cells and the reorganization of the auditory epithelium following the treatment. Our second Aim is characterize the response of inner ear neurons to the presence of new hair cells. Our third Aim is to test the hypothesis that transduction channels at the tips of stereocilia of new hair cells are functional, and determine the influence of Mathl gene therapy on hearing thresholds in deaf guinea pigs. The novel and feasible experiments we propose will help to characterize and enhance hair cell regeneration induced by the Mathl transgene in the mature guinea pig cochlea. The work we describe will bring the innovative field of hair cell regeneration research closer to clinical applicability.